Functionally Relevant Diversity of Closely Related Nitrospira in Activated Sludge

Home , Lithoautotroph

The ISME Journal (2015) 9, 643–655 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Functionally relevant diversity of closely related Nitrospira in activated sludge

Christiane Gruber-Dorninger1, Michael Pester1,3, Katharina Kitzinger1, Domenico F Savio1,4, Alexander Loy1, Thomas Rattei2, Michael Wagner1 and Holger Daims1 1Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria and 2Division of Computational Systems Biology, Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria

Nitrospira are chemolithoautotrophic nitrite-oxidizing bacteria that catalyze the second step of nitrification in most oxic habitats and are important for excess nitrogen removal from sewage in wastewater treatment plants (WWTPs). To date, little is known about their diversity and ecological niche partitioning within complex communities. In this study, the fine-scale community structure and function of Nitrospira was analyzed in two full-scale WWTPs as model ecosystems. In Nitrospira-specific 16S rRNA clone libraries retrieved from each plant, closely related phylogenetic clusters (16S rRNA identities between clusters ranged from 95.8% to 99.6%) within Nitrospira lineages I and II were found. Newly designed probes for fluorescence in situ hybridization (FISH) allowed the specific detection of several of these clusters, whose coexistence in the WWTPs was shown for prolonged periods of several years. In situ ecophysiological analyses based on FISH, relative abundance and spatial arrangement quantification, as well as microautoradiography revealed functional differences of these Nitrospira clusters regarding the preferred nitrite concentration, the utilization of formate as substrate and the spatial coaggregation with ammonia-oxidizing bacteria as symbiotic partners. Amplicon pyrosequencing of the nxrB gene, which encodes subunit beta of nitrite oxidoreductase of Nitrospira, revealed in one of the WWTPs as many as 121 species-level nxrB operational taxonomic units with highly uneven relative abundances in the amplicon library. These results show a previously unrecognized high diversity of Nitrospira in engineered systems, which is at least partially linked to niche differentiation and may have important implications for process stability. The ISME Journal (2015) 9, 643–655; doi:10.1038/ismej.2014.156; published online 22 August 2014

Introduction Freitag et al., 2005; Off et al., 2010). Within this genus, six phylogenetic main lineages have been Nitrification, the sequential oxidation of ammonia to described that occur in a wide range of habitats nitrite to nitrate, is a key nitrogen cycling process in (Daims et al., 2001a; Lebedeva et al., 2008; Off et al., oxic ecosystems and an important step of biological 2010). In addition, we recently detected an immense wastewater treatment. Nitrification is a two-step diversity of coexisting Nitrospira from these six and process carried out by chemolithoautotrophic several novel lineages in various soils by amplicon ammonia-oxidizing bacteria and archaea (AOB, pyrosequencing the nxrB gene, which codes for AOA) and nitrite-oxidizing bacteria (NOB). subunit beta of nitrite oxidoreductase and is a useful In the environment and in wastewater treatment functional and phylogenetic marker for Nitrospira plants (WWTPs), the most diverse and often (Pester et al., 2013). predominant known NOB are mainly uncultured Most Nitrospira in WWTPs are affiliated to the members of the genus Nitrospira (Burrell et al., main lineages I or II (Daims et al., 2001a; Maixner 1998; Juretschko et al., 1998; Daims et al., 2001a; et al., 2006). These two lineages can coexist in the same full-scale WWTPs or lab-scale reactors Correspondence: H Daims, Division of Microbial Ecology, (Schramm et al., 1998; Maixner et al., 2006; Park Department of Microbiology and Ecosystem Science, University and Noguera, 2008) apparently without competitive of Vienna, Althanstrae 14, 1090 Vienna, Austria. E-mail: [email protected] exclusion and based on ecological niche partition- 3Current address: Department of Biology, University of Konstanz, ing. Past research showed that lineage I Nitrospira Konstanz, Germany. preferred a higher nitrite concentration than lineage 4Current address: Division of Biotechnology and Microbiology, II (Maixner et al., 2006), but lineage II Nitrospira Institute of Chemical Engineering, Vienna University of Techno- logy, Vienna, Austria. were better adapted to elevated levels of dissolved Received 28 February 2014; revised 28 June 2014; accepted 18 oxygen (Park and Noguera, 2008). However, it July 2014; published online 22 August 2014 remains unclear whether the main lineages Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 644 represent the functionally relevant scale of until DNA extraction. Additional sludge aliquots Nitrospira diversity. If these lineages comprise an were fixed immediately upon arrival in the lab in additional diversity of more closely related clades, 3% (v v À 1) formaldehyde in 1 Â phosphate-buffered which still differ in ecophysiological traits and can saline for 2 h at 4 1C and were stored in 96% coexist by niche partitioning, a higher phylogenetic ethanol:1 Â phosphate-buffered saline (1:1) at resolution than the main Nitrospira lineages may À 20 1C. For fluorescence in situ hybridization- be needed to understand the biology of nitrite microautoradiography (FISH-MAR) analyses, sludge oxidation. was shipped to the laboratory at 4 1C and immedi- For other bacterial groups, highly diverse ately processed upon arrival. communities of closely related, coexisting members were reported based on marker gene or meta- genomic sequence analyses (e.g., Acinas et al., DNA extraction, clone library construction, Sanger 2004; Cuadros-Orellana et al., 2007; He et al., sequencing and phylogenetic analyses 2007; Woebken et al., 2008; Denef et al., 2010; DNA was extracted from activated sludge sampled Albertsen et al., 2012; Ngugi and Stingl, 2012). in February 2009 and October 2010 from WWTP However, the functional differentiation of closely Vetmed and in March 2011 from WWTP Ingolstadt related uncultured microbes has not been investi- using the Powersoil DNA Isolation Kit (MO BIO gated directly in the source communities by in situ Laboratories, Carlsbad, CA, USA) and following the techniques. Here, we tackled this issue by combin- manufacturer’s instructions. ing phylogenetic analyses of Nitrospira 16S rRNA Nitrospira partial 16S rRNA genes were genes with single-cell in situ tools. This cultivation- PCR-amplified using primers 8F (Hicks et al., independent approach allowed us to investigate the 1992; Juretschko et al., 1998) and Ntspa1158R fine-scale community structure and ecophysiology (Maixner et al., 2006) according to the protocol of of uncultured Nitrospira, below the phylogenetic Maixner et al. (2006) with the following modifica- level of the main lineages, in two full-scale nitrify- tions: initial denaturation for 5 min at 95 1C, primer ing WWTPs. Additionally, the Nitrospira diversity annealing at 58 1C and final elongation for 10 min at in one WWTP was explored in great depth by 72 1C. PCR products were cloned by using the TOPO amplicon pyrosequencing the nxrB gene. TA cloning system (Invitrogen, Carlsbad, CA, USA), and Sanger sequencing was performed by using the BigDye Terminator Cycle Sequencing Kit v.3.1 Materials and methods and an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the Sampling and fixation of activated sludge manufacturer’s instructions. Activated sludge was sampled from an aerated, Nitrospira 16S rRNA sequences were phylogeneti- nitrifying activated sludge basin (tank no. 2) of the cally analyzed by using the ARB software (Ludwig full-scale WWTP of the University of Veterinary et al., 2004) and a sequence database containing 345 Medicine, Vienna, Austria (‘WWTP Vetmed’) in almost full-length (41350 nucleotides) reference March, April and July 2004, May and June 2007, sequences from the phylum Nitrospirae, which had November 2008, February and October 2009 and in been retrieved from GenBank. Sequence alignments July and October 2010. This WWTP consists of two generated by ARB were manually refined. To check continuously operated activated sludge tanks, each for chimeric sequences, each sequence was with a volume of 254 m3. The influent wastewater separated into two equal parts. The 50 and 30 parts composition, including the N-load, varies with the were independently added to a Nitrospira reference amounts of animal feces and other sewage. The tree by using the parsimony interactive tool of ARB effluent of WWTP Vetmed is subsequently treated by without changing the overall tree topology, and the main municipal WWTP of the city of Vienna. sequences showing placement of the two parts in Nitrifying activated sludge samples were also taken different Nitrospira lineages were discarded as from a sequencing batch reactor, which treats reject probably chimeric. Phylogenetic trees of Nitrospira water from sludge dewatering after anaerobic diges- lineages I and II were calculated using maximum tion, at the municipal full-scale WWTP of Ingolstadt likelihood inference (AxML) (Stamatakis et al., (Germany) in October 2007, November 2008, 2002), neighbor joining (ARB implementation, October 2009 and March 2011. The reject water Felsenstein distance correction model) and treatment stage of this WWTP consists of three maximum parsimony within the PHYLIP package sequencing batch reactors, which together treat up (Felsenstein 1989) with a 50% conservation filter for to 450 m3 wastewater per day with an ammonium the phylum Nitrospirae that included 1409 unam- þ load of 450 kg NH4 -N per day. Each sequencing biguously aligned nucleotide positions. batch reactor is intermittently aerated for N-elimina- tion by nitrification and denitrification. All samples were taken from the same sequencing batch reactor. Probe design, FISH, microscopy and digital image analysis Activated sludge was collected by centrifugation To design 16S rRNA-targeted oligonucleotide probes (11000 r.p.m., 10 min, 4 1C) and frozen at À 20 1C specific for single phylogenetic clusters within

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 645 Nitrospira lineage I or II, we screened the Nitrospira incubated with one of the substrates [14C]glucose, 16S rRNA sequences from the two WWTPs for probe [14C]formate, [14C]acetate, [14C]pyruvate and [14C]L- target regions with cluster-specific single-nucleotide valine (Hartmann Analytic, Braunschweig, Ger- polymorphisms. The new probes should distinguish many) in the presence of 1 mM nitrite or 1 mM the Nitrospira clusters, which occurred in the same ammonium or without the addition of any inorganic

WWTP, from each other rather than from Nitrospira electron donor. Autotrophic CO2 fixation was stu- 14 À in other habitats or from other bacteria. To compen- died by incubating sludge with H CO3 (Hartmann sate for a possible lack of specificity outside the Analytic, Braunschweig, Germany) in the presence genus Nitrospira, these probes were combined with of 1 or 0.1 mM nitrite, 1 mM ammonium or without established specific probes targeting the entire the addition of any inorganic electron donor. Nitrospira lineages I or II or the whole genus, Incubations with dead, formaldehyde-fixed sludge respectively, which were labeled with a different were performed to check for adsorption of the fluorochrome (Supplementary Table S1). The opti- labeled substrates to Nitrospira. Further details are mal hybridization and washing conditions for each provided in Supplementary Text S1. Incubated new probe were inferred from probe dissociation sludge was fixed in 1.5% (v v À 1) formaldehyde in curves with the targeted 16S rRNA molecules as 1 Â phosphate-buffered saline overnight at 4 1C. described elsewhere (Daims et al., 1999) by using FISH-MAR was performed as described previously Clone-FISH (Schramm et al., 2002) and the image (Lee et al., 1999). For each experiment, at least 30 analysis software daime (Daims et al., 2006a). The (up to 164) microcolonies of each analyzed Nitros- specificity of the new probes within the Nitrospira pira phylogenetic cluster were inspected for the communities was confirmed in FISH experiments presence or absence of a MAR signal and counted. with non-target Nitrospira 16S rRNA (also obtained w2 Tests of significance were performed between by Clone-FISH) with one nucleotide mismatch at the incubations involving the same carbon source and probe binding sites. Probes were 5’ labeled with the Nitrospira cluster by using IBM SPSS Statistics 21.0. fluorochromes Cy3, Cy5 or FLUOS. Unlabeled We could not exclude the possibility that environ- competitor probes (Supplementary Table S1) and mental parameters such as temperature and pH, double-labeled probes (to increase fluorescence which were kept constant in all incubations, were intensity) (Stoecker et al., 2010) were used when suboptimal for some of the studied Nitrospira necessary. All probes were obtained from Thermo phylogenetic clusters and thus caused different Hybaid (Ulm, Germany). levels of the metabolic activity of these NOBs. FISH was performed according to Daims et al. Therefore, the results of the different incubations (2005) with hybridization times of 2–16 h (fractions of MAR-positive microcolonies) were (Supplementary Table S1). Images of probe-con- compared only for the same Nitrospira cluster but ferred fluorescence were recorded by using a not between different clusters. confocal laser scanning microscope (510 Meta; Zeiss, Oberkochen, Germany). The relative abundance (biovolume fraction) of specific Nitrospira Long-term incubation clusters (compared with all Nitrospira detected by Activated sludge from WWTP Vetmed (sampled in probe Ntspa662, to all lineage II Nitrospira detected November 2010) was diluted 1:4 in filter-sterilized by probe Ntspa1151 or to all lineage I Nitrospira (0.2 mm) sludge supernatant. Sludge aliquots of detected by Ntspa1431) was measured by quantita- 50 ml were incubated in 200 ml glass bottles sealed tive FISH as described elsewhere (Daims and with aluminum caps with a polytetrafluoroethylene Wagner, 2007) and by using the daime software. In butyl septum (Carl Roth, Karlsruhe, Germany) in the situ spatial arrangement patterns of Nitrospira and dark at 20 1C and in the presence of 0.05 or 1 mM AOB were quantified as described previously nitrite. These nitrite concentrations support the (Daims et al., 2006a). Briefly, the pair cross-correla- growth of Nitrospira isolates and enrichments tion function was estimated, by image analysis, for a (Ehrich et al., 1995; Spieck et al., 2006), and in range of mm distances between two FISH probe- preliminary experiments using these nitrite concen- 14 À defined target groups. For each distance, the pair trations and H CO3 , we observed MAR-positive cross-correlation function indicates the likelihood Nitrospira microcolonies in sludge from WWTP that the two analyzed groups are within this Vetmed (data not shown). All incubations were distance compared with a random arrangement of performed in duplicates. During the 51 days of cells. Further details of FISH and image analysis are incubation, the nitrite concentration was frequently provided in Supplementary Text S1. checked colorimetrically (Supplementary Text S1) and nitrite was replenished regularly by using peristaltic pumps. To avoid carbon depletion, FISH and microautoradiography 10 mM of NaHCO3 was added on days 27 and 47. Substrate uptake experiments were performed with The pH was measured every 1–2 days and adjusted living activated sludge from WWTP Ingolstadt to 7.6 by adding HCl when necessary. To prevent sampled in March 2011. To explore the use of nitrate accumulation and possible trace element organic substrates by Nitrospira, sludge was or cofactor depletion, culture supernatant was

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 646 replaced by filter-sterilized activated sludge super- clusters (ranging from 95.8% to 99.6%). In total, 11 natant every 7–10 days. Air was pumped into the clusters were defined, some of which contained headspace for 2 min every 4 h by using an aquarium only sequences from one of the two studied WWTPs pump (Eheim 106001; Eheim, Deizisau, Germany), (Figure 1). One lineage I (Ingolstadt clone 43) and and the cultures were continuously shaken at one lineage II sequence (Vetmed clone 09-19) did not 90 r.p.m. to ensure sufficient aeration. Samples were group with other sequences from this study taken at five time points and were formaldehyde- (Figure 1). Several clusters contained also Nitrospira fixed as described above. sequences retrieved in other studies from WWTPs, bioreactors and iron-oxidizing biofilm (Figure 1 and Supplementary Table S2). Amplicon pyrosequencing of Nitrospira nxrB The nxrB genes of Nitrospira were PCR amplified from WWTP Ingolstadt-activated sludge (sampled in In situ detection of closely related Nitrospira November 2008). DNA extraction, PCR with primer To confirm that the Nitrospira sequence clusters set nxrB169f/nxrB638r, 454 amplicon sequencing represented autochthonous community members in and bioinformatics analyses were performed as the two WWTPs and did not result from transiently described previously (Pester et al., 2013). Approxi- present cells, naked DNA or PCR and sequencing mate species-level operational taxonomic units errors, FISH experiments were carried out to detect (OTUs) were formed by clustering the nxrB reads the Nitrospira clusters in situ. For this purpose, new with 95% nucleotide sequence identity as cutoff 16S rRNA-targeted oligonucleotide probes were (Pester et al., 2013). Representative sequences from designed based on the sequences in each cluster selected OTUs were added to an nxrB reference tree (Supplementary Table S1). Owing to the high 16S (Pester et al., 2013) by using the parsimony inter- rRNA sequence identities between the different active tool of ARB or the maximum likelihood-based Nitrospira clusters (see above), the design of evolutionary placement algorithm (Berger et al., cluster-specific probes had to rely on SNPs at the 2011), respectively, without changing the overall probe binding sites to distinguish the clusters. As tree topology. The reference tree was based on the unique SNPs were rare, we could not design a tree of Pester et al. (2013) but contained additionally universally specific probe for each cluster. Instead, the two paralogous nxrB copies of Nitrospina the new probes specifically distinguished the gracilis (Lu¨ cker et al., 2013) as outgroup. clusters found in the same WWTP but might have been unspecific if applied to the respective other WWTP or to samples from other environments. Accession numbers Therefore, each new probe was applied to only one The 16S rRNA gene sequences obtained in this of the WWTPs analyzed in this study (Figure 1) and study have been deposited at GenBank under use of these probes on other samples is not accession numbers KJ480818–KJ480936. The nxrB recommended. sequences were submitted to the Sequence Read In WWTP Vetmed, Nitrospira clusters Id, Ie, IIa, Archive (SRA) at GenBank under the accession IIb and IIc were specifically detected by FISH number SRA047303. (Figures 2a–c), demonstrating that these Nitrospira indeed coexisted in the WWTP. Notably, probe Results Ntspa1012 targeting cluster Ie has no base mismatch to Vetmed clone 09-2, which is outside cluster Ie Nitrospira 16S rRNA gene diversity in two WWTPs (Supplementary Table S2) but could thus not be By cloning and Sanger sequencing, partial distinguished from cluster Ie by FISH using this sequences of Nitrospira 16S rRNA genes (1126– probe. In WWTP Ingolstadt, clusters Ia, Ib and Ig 1132 bp) were retrieved from WWTP Vetmed in were detected by the respective FISH probes February 2009 (n ¼ 40) and October 2010 (n ¼ 22) (Figures 2d and e). Cluster IIa was detected in and from WWTP Ingolstadt in March 2011 (n ¼ 58) WWTP Ingolstadt by probe Ntspa1151 targeting (Supplementary Table S2). Based on these sequence Nitrospira lineage II (no specific probe was applied data, phylogenetic clusters within the main Nitros- because cluster IIa were the only lineage II-related pira lineages I and II were defined. The criterion Nitrospira found in this WWTP; Figure 1). The other used for clustering was that all members of the same Nitrospira clusters (Figure 1) were not detected by cluster must form a monophyletic group in at least FISH (for details see Supplementary Text S1). All one phylogenetic tree (ideally in all trees calculated Nitrospira clusters identified in situ hybridized also by different treeing methods). The smallest identi- to the Nitrospira genus-specific probe Ntspa662 and, fied monophyletic groups were defined as separate except cluster Ig, to the respective group-specific clusters to maximize the phylogenetic resolution of probes targeting Nitrospira lineages I or II the grouping. Sequences within the same cluster (Supplementary Table S1 and Figure 2). were highly similar to each other, with identities All Nitrospira formed spherical or irregularly ranging from 98.6% to 99.9%, but high sequence shaped microcolonies as typically observed for these identities were also observed between different NOB in activated sludge (Juretschko et al., 1998;

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 647 cluster Ia (2) Ingolstadt:Ntspa194

cluster Ib (18) Ingolstadt:Ntspa1131

cluster Ic (9)

EBPR plant clone Skagenf5, DQ640657 branch supported aerobic P−removal ecosystem clone PHOS−HE34, AF314422 cluster Id (9) Vetmed: Ntspa1002 branch not supported maximum likelihood maximum parsimony cluster Ie (13) Vetmed: Ntspa1012 tree tree nitrit-oxidizing bioreactor clone RC19, Y14638 nitrite-oxidizing bioreactor clone, Y14641 nitrite-oxidizing bioreactor clone RC7, Y14640 oxytetracycline production waste clone MY 54, JN245677 activated sludge clone GD 1−73, KC551598 EBPR sludge clone 71, DQ413129 cluster If (2) soil clone DM2−146, KC172221 biofilm clone, AB117711 farm soil clone AKYH1008, AY921886 wastewater nitrifying biofilm clone, JQ936547 rice field soil clone AP19, AY921494 cluster Ig (3) Ingolstadt: Ntspa451

Ingolstadt WWTP clone 43, KJ480921 cluster IIa (34) Vetmed: Ntspa256

cluster IIb (12) Vetmed: Ntspa175

estrogen−degrading membrane bioreactor clone M1−5, EU015101

cluster IIc (23) Vetmed: Ntspa195

cluster IId (4) fluidized bed reactor clone o9, AJ224042 Cretan margin sediment clone, EU374034 Vetmed WWTP clone 09-19 drinking water distribution system simulator clone, AY328760

Nitrospira lineage II clones (4) Nitrospira moscoviensis, X82558 Nitrospira lineage II clones (8)

Nitrospira lineage II clones (5) Nitrospira enrichment culture clone Ga9−4, HM485587 1% Figure 1 Phylogenetic analysis of partial Nitrospira 16S rRNA gene sequences retrieved from WWTPs Vetmed and Ingolstadt. The neighbor joining tree depicts the identified phylogenetic clusters from Nitrospira lineages I and II, with clusters found in WWTP Vetmed colored blue, clusters found in WWTP Ingolstadt colored red and clusters found in both WWTPs colored blue and red, respectively. Clusters also containing Nitrospira sequences from other environments are marked with black in addition to the colors, whereas groups that fully consist of sequences from other sources are drawn completely black. Numbers within parentheses behind cluster names indicate the numbers of sequences in the clusters. The names of FISH probes used to detect the different clusters in situ are indicated together with the name of the respective WWTP and in the matching color. Horizontal gray bars, which link the clusters and probe names, illustrate that each probe was designed for and applied to only one Nitrospira community (in the respective WWTP). Circles on tree branches indicate the support by other treeing methods of the respective monophyletic clusters. Sequences of Nitrospira lineage IV were used as outgroup. The scale bar indicates 1% estimated sequence divergence. The accession numbers of the sequences in each cluster are listed in Supplementary Table S2.

Daims et al., 2001a) (Figure 2). As none of these genomes of ‘Ca. N. defluvii’ (Lu¨ cker et al., 2010) microcolonies hybridized to more than one of the and N. moscoviensis (unpublished data). cluster-specific probes in each WWTP (Figure 2), we can largely exclude the possibility that the sequence clusters (Figure 1) might represent multi- Stable coexistence of closely related Nitrospira: an ple 16S rRNA gene copies of very few Nitrospira indication of niche partitioning? strains. This result of FISH is in agreement with FISH using the newly designed high-resolution the presence of only one rrn operon in the probes revealed that in each WWTP, the closely

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 648

Figure 2 Closely related Nitrospira clusters detected by FISH in WWTPs Vetmed and Ingolstadt. All scale bars depict 10 mm. (a) Clusters IIa (yellow) and IIc (cyan) in WWTP Vetmed (probe Ntspa195 in blue, Ntspa256 in red and Ntspa1151 in green). (b) Clusters IIa and IIc (both cyan) and IIb (yellow) in WWTP Vetmed (probe Ntspa195 and Ntspa256 in blue, Ntspa175 in red and Ntspa1151 in green). (c) Clusters Id (purple) and Ie (yellow) in WWTP Vetmed (probe Ntspa1002 in blue, Ntspa1012 in green and Ntspa1431 in red). (d) Clusters Ia (yellow) and Ib (purple) in WWTP Ingolstadt (probe Ntspa1131 in blue, Ntspa194 in green and Ntspa1431 in red). (e) Cluster Ig (purple) in WWTP Ingolstadt (probe Ntspa451 in red, Ntspa1431 and Ntspa1151 in green, and Ntspa662 in blue). Note that cluster Ig was missed by the lineage I-specific probe Ntspa1431.

related Nitrospira clusters apparently coexisted fine-scale community structure within lineage I. during prolonged periods for which a sparse set of Throughout the experiment, the relative abundance activated sludge samples was available (B6 years of cluster Ie was significantly higher in the incuba- for WWTP Vetmed and B4 years for WWTP tions with 1 mM nitrite (Figure 3a). The situation was Ingolstadt) (data not shown). Interestingly, the co- reversed for cluster Id, whose relative abundance occurrence of the lineage II Nitrospira clusters in the was higher in the incubations with 0.05 mM nitrite samples from WWTP Vetmed was accompanied by between days 14 and 31 of the experiment. Subse- pronounced shifts in the lineage II Nitrospira quently, cluster Id declined in all incubations community composition (Supplementary Text S1 (Figure 3b). and Supplementary Figure S1). Observation by non-quantitative FISH revealed no The detection of a diverse community of stably obvious effect of the nitrite concentrations on the coexisting, closely related Nitrospira raised the different lineage II clusters. During all incubations question of whether these organisms are function- and in the original sludge sampled for this experi- ally different and coexist by ecological niche ment, cluster IIc was predominant and easily partitioning. To address this question, we took detectable by FISH, whereas clusters IIa and IIb advantage of the unique possibility to detect, were encountered only sporadically in some of the distinguish and quantify the closely related Nitros- inspected fields of view (data not shown). As the pira in situ and to monitor their substrate utilization low abundance of clusters IIa and IIb was close to by FISH-MAR. the detection limit of FISH, quantitative FISH would not be precise enough for measuring small abundance shifts of these extremely rare Nitrospira. Preference for different nitrite concentrations We carried out a long-term incubation experiment with activated sludge to test the effects of two Autotrophic and mixotrophic carbon utilization ambient nitrite concentrations (0.05 and 1 mM)on Another potentially niche-defining property of different Nitrospira clusters. This experiment was Nitrospira could be the utilization of simple organic conducted with sludge from WWTP Vetmed, which substrates in addition to nitrite and CO2 (mixotro- contained different clusters within both Nitrospira phy) (Watson et al., 1986; Daims et al., 2001a; lineages I and II (Figure 1). Lu¨ cker et al., 2010). To test whether closely related Interestingly, quantitative FISH revealed distinct Nitrospira differed in their mixotrophic potential effects of the two nitrite concentrations on the and substrate spectrum, the uptake by lineage I

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 649

70 70 1 mM nitrite, biological replicate 1 1 mM nitrite, biological replicate 2 60 60 0.05 mM nitrite, biological replicate 1 0.05 mM nitrite, biological replicate 2 50 50 Original sludge 40 40

30 30

20 20 to total lineage I (%) 10 10 Biovolume fraction relative 0 0 0 14 27 31 47 51 0 14 27 31 47 51 Time (d) Time (d) Figure 3 Relative abundance shifts of Nitrospira lineage I clusters from WWTP Vetmed during incubation with two different nitrite concentrations. All incubations were performed in duplicates (biological replicates 1 and 2), and two technical replicates per time point were analyzed by qFISH. (a) Relative abundance (biovolume fraction) of cluster Ie, compared with total Nitrospira lineage I, at different time points during the incubation. The relative abundance of cluster Ie was significantly higher (Mann–Whitney U-test: Po0.001; t-test: Po0.001) in samples from the high nitrite incubations taken between days 14 and 51. Symbols are as in panel b. (b) Biovolume fraction of cluster Id, relative to total Nitrospira lineage I, at different time points during the incubation. The relative abundance of cluster Id was significantly higher (Mann–Whitney U-test: Po0.001; t-test: Po0.001) in samples from the low nitrite incubations taken between days 14 and 31.

clusters of radiolabeled formate, acetate, pyruvate, - + HCO3 , 1 mM NH4 2 mM formate - - + L-valine and glucose was monitored by FISH-MAR. HCO3 , 1 mM NO2 2 mM formate, 1 mM NH4 HCO -, 0.1 mM NO - 2 mM formate, 1 mM NO - Based on the genomic inventory of ‘Ca. N. defluvii’ 3 2 2 (a lineage I member), we hypothesized that these 100 substrates might be used by lineage I Nitrospira (Lucker et al., 2010). These experiments were ¨ 75 *** carried out with sludge from WWTP Ingolstadt as *** lineage I was more diverse in this WWTP than in *** WWTP Vetmed (Figure 1). Additional incubations 50 with 14C-labeled bicarbonate instead of organics tested the autotrophic activity of the different lineage I Nitrospira. All incubations were performed in 25 * the presence or absence of an inorganic energy source (nitrite or ammonium). The experiments with *** * Fraction of MAR-positive microcolonies (%) 0 ammonium were included because Nitrospira might Cluster Ia Cluster Ib Cluster Ig be best adapted to the (unknown) local nitrite Figure 4 Incorporation of 14C-labeled bicarbonate and formate by concentrations close to nitrite-producing AOB Nitrospira lineage I clusters from WWTP Ingolstadt. Fractions of (Maixner et al., 2006). MAR-positive FISH-identified microcolonies (nX30 for each All in situ detectable Nitrospira lineage I clusters cluster) were counted after the different incubation experiments. 2 incorporated bicarbonate and formate, but the Significantly different fractions (according to w -tests on values obtained for one cluster and carbon source) are indicated by fractions of MAR-positive microcolonies varied *Po0.05 or ***Po0.001. substantially between clusters and conditions. Cluster Ib was most active with the highest fraction of MAR-positive microcolonies (Po0.001) in all formate-labeled cluster Ig microcolonies was highest experiments. The autotrophic activity of clusters Ia with 1 mM nitrite (Figure 4). Cluster Ia used formate and Ib was not influenced by the incubation most actively in the incubation amended with conditions, whereas the percentage of MAR-positive ammonium (Figure 4). Finally, only cluster Ib used cluster Ig microcolonies was significantly higher in formate efficiently as the sole substrate (Figure 4 and the presence of 1 mM nitrite than in the other Supplementary Figure S2). incubations with 14C-bicarbonate (Figure 4). No No Nitrospira lineage I cluster in WWTP Ingol- Nitrospira assimilated bicarbonate in the absence stadt utilized pyruvate, glucose, acetate or valine, of nitrite and ammonium (data not shown). whereas uptake of the latter three substrates by other Interestingly, formate utilization was more hetero- bacteria was observed. The lack of uptake by geneous than CO2 fixation. When incubated with Nitrospira may be due to genetic differences 1mM nitrite, formate incorporation by cluster Ib was of the analyzed lineage I clusters compared with significantly lower than with 1 mM ammonium or ‘Ca. N. defluvii’ and awaits explanation until more formate alone (Figure 4). In contrast, the fraction of Nitrospira genomes become available.

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 650 Spatial arrangement patterns comparing the three Nitrospira lineage I clusters In activated sludge flocs and sessile biofilms, with each other (Supplementary Text S1 and Nitrospira coaggregate with AOB because nitrifiers Supplementary Figure S3). live in mutualistic symbiosis where ammonia The spatial distribution between Nitrospira oxidizers produce nitrite, which is consumed and targeted by probe Ntspa1151 (specific for Nitrospira detoxified by NOB (Juretschko et al., 1998; Stein and lineage II; Supplementary Table S1) and the Arp, 1998; Daims et al., 2001a; Almstrand et al., Ncom1025-targeted AOB was also quantified, but 2013). Previously, we showed that lineage I no coaggregation was observed (data not shown). Nitrospira occurred closer to AOB microcolonies than members of lineage II, probably because small-scale nitrite concentration gradients formed In-depth analysis of Nitrospira richness in WWTP around AOB, and Nitrospira lineages I and II settled Ingolstadt along those gradients according to their different The Nitrospira richness in WWTP Ingolstadt was nitrite optima (Maixner et al., 2006). Here we assessed in more depth by amplicon pyrosequencing extended our earlier study, which had analyzed of nxrB.Intotal,9549Nitrospira nxrB reads passed the Nitrospira only at the level of main lineages, by quality screening criteria described in Pester et al. quantifying the in situ spatial arrangement of (2013). Clustering of these reads using a nucleotide coexisting lineage I members relative to AOB and sequence identity threshold of 95%, which is the to each other. Different localization patterns might suggested species threshold for Nitrospira nxrB indicate niche-separating features of these closely (Pester et al., 2013), resulted in 121 Nitrospira related Nitrospira. These analyses were carried out species-level OTUs that apparently coexisted in with sludge from WWTP Ingolstadt, where the the WWTP. As suggested by a high Good’s coverage different Nitrospira clusters were more equally (Good, 1953) of 0.996, the Nitrospira richness in the abundant and AOB detected by available AOB- amplicon library was almost completely covered by targeted FISH probes were much more abundant the applied sequencing approach. The ACE and than in WWTP Vetmed (data not shown). As the Chao1 nonparametric richness estimators predicted AOB-targeted probes (Supplementary Table S1) 159 (95% confidence interval 140–195) and 147 unexpectedly showed inconsistent hybridization (95% confidence interval 132–180) species-level patterns (Supplementary Text S1), only the signal OTUs, respectively. As typical 454 pyrosequencing of the AOB-targeted probe Ncom1025 was evaluated. errors can cause overestimation of microbial diversity We detected a strong and highly specific tendency (Kunin et al., 2010), our analysis pipeline combined of Nitrospira cluster Ig to coaggregate with the several approaches for bias reduction including Ncom1025-targeted AOB over a distance range from sequence quality screening and filtering, frameshift 9to54mm (Figure 5a). Intriguingly, a completely detection, manual sequence curation and chimera different pattern was observed for Nitrospira clus- exclusion (Pester et al., 2012, 2013). Thus, the 55 ters Ia and Ib that were either randomly distributed OTUs, which were represented by only one or two relative to these AOB (cluster Ia; Figure 5b) or were quality-controlled 454 reads very likely represent rare even negatively correlated with Ncom1025-targeted Nitrospira species, but we cannot exclude the possi- AOB up to a distance of 42 mm (cluster Ib; Figure 5c). bility that some of these rare OTUs result from Different localization patterns were also detected by undetected 454 sequencing errors in single reads.

Cluster Ig vs. Cluster Ia vs. Cluster Ib vs. Ncom1025-targeted AOB Ncom1025-targeted AOB Ncom1025-targeted AOB ) 2 2 2 2

0 0 0

-2 -2 -2

-4 -4 -4

-6 -6 -6 Pair cross-correlation (log

0 10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Distance (µm) Figure 5 (a–c) Spatial arrangement of Nitrospira lineage I clusters relative to probe Ncom1025-targeted AOB in WWTP Ingolstadt. The

pair cross-correlation is plotted as a function of distance between the NOB and AOB. Pair cross-correlation values above one (log2 ¼ 0, horizontal line) indicate coaggregation, values below one (log2 ¼ 0) indicate a negative spatial correlation and values not significantly different from one (log2 ¼ 0) indicate random distribution at the respective distance. Continuous lines depict means calculated from all analyzed images, and error bars show 95% confidence intervals.

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 651 Only six of the 121 species-level OTUs were Activated sludge offers microhabitats due to the high in relative abundance (45%) and together spatial heterogeneity of flocs that lead to strong represented approximately 73% of all nxrB gradients of electron donors and acceptors (e.g., sequence reads (Figure 6). As the genomic copy Schramm et al., 1999; Maixner et al., 2006). This and numbers of nxrB range from 2 to 6 in known temporal variations of environmental parameters Nitrospira species (Pester et al., 2013), the quanti- during the operation of WWTPs (e.g., Daims et al., tative composition of the amplicon library may be 2001a) may support the coexistence of NOB strains influenced by different nxrB copy numbers in with different ecophysiological adaptations (niche Nitrospira genomes from different OTUs. However, partitioning). For various microorganisms other than this possible bias would not explain the whole range NOB, ecological niche partitioning of closely related of observed difference in relative abundance and strains has been inferred from covariation of the we conclude that the Nitrospira community compo- strains with environmental parameters or other sition was highly uneven (Figure 6). Phylo- organisms (e.g., Coleman and Chisholm, 2007; genetic analyses were conducted with represen- Fuhrman and Steele, 2008; Hunt et al., 2008; tative sequences from the species-level OTUs Koeppel et al., 2008; Zo et al., 2008; Connor et al., (Supplementary Table S3 and Supplementary 2010; Denef et al., 2010; Brown et al., 2012). Niche Figure S4). In total, 79 (65%) of the OTUs were partitioning of closely related strains was also closely related to Nitrospira lineages I or II that were suggested by differential pigmentation of photo- originally defined based on 16S rRNA gene phylo- trophs (e.g., Acinas et al., 2009; Haverkamp et al., genies (Daims et al., 2001a) and are also resolved in 2009), by differences in gene content, protein nxrB-based phylogenetic trees (Pester et al., 2013). expression and metabolites (Denef et al., 2010; Four OTUs of low abundance were tentatively Pena et al., 2010), by in silico evolutionary simula- assigned to a new ‘WWTP Ingolstadt 454 lineage’ tions (Koeppel et al., 2008; Connor et al., 2010; based on their position in the genus Nitrospira, but Becraft et al., 2011) and by comparisons of isolates outside established lineages in the nxrB reference (e.g., Hunt et al., 2008; Acinas et al., 2009; tree (Supplementary Figure S4 and Supplementary Malmstrom et al., 2010; Pena et al., 2010; Verbeke Table S3). The phylogenetic position of 38 OTUs et al., 2011). All these approaches yielded important within the genus Nitrospira was ambiguous insight into functional traits of closely related (Supplementary Table S3), and some of these microorganisms. However, actual phenotypic differ- unassigned OTUs were abundant in the nxrB ences have mainly been demonstrated for cultured amplicon library (Figure 6). isolates. In contrast, ecophysiological studies of specific, closely related, uncultured microorganisms directly in environmental samples have to our Discussion knowledge not been undertaken yet. Filling this research gap is important because isolating a In two full-scale WWTPs, application of the collection of closely related strains of slow-growing full-cycle rRNA approach revealed an unexpect- microbes from a sample is an enormous challenge, edly high diversity of closely related Nitrospira and observations made with pure cultures or that coexisted over several years. Moreover, in artificial mixtures of isolates do not reflect the oneoftheWWTPsasmanyas121species-level situation in complex communities. Here, the detec- OTUs of Nitrospira were detected by nxrB pyrose- tion of a high diversity of uncultured Nitrospira by quencing, highlighting the enormous amount of FISH with SNP-discriminating, rRNA-targeted apparently coexisting Nitrospira that escape or probes paved the way for first in situ experiments avoid competitive exclusion in these man-made to elucidate the ecophysiology and coexistence of systems. closely related Nitrospira.

1600 Lineage I 1400 35 Lineage II Related to lineage I 1200 30 Related to lineage II 25 WWTP Ingolstadt 454 lineage 1000 Unassigned Nitrospira 20 800 15 600 10 Number of reads 400 5 0 200 30 40 50 60 70 80 90 100 110 120 0 10 20 30 40 50 60 70 80 90 100 110 120 OTU abundance rank Figure 6 Relative abundances of species-level OTUs detected in WWTP Ingolstadt by Nitrospira nxrB amplicon pyrosequencing. Colors indicate the phylogenetic affiliation of the OTUs with the established Nitrospira lineages I and II, and with the new ‘WWTP Ingolstadt 454 lineage’ (see also Supplementary Table S3 for details).

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 652 Niche differentiation of closely related Nitrospira phylogenetic level studied here. We cannot exclude This study provides multiple lines of evidence for that additional specific coaggregation existed also ecological niche partitioning of coexisting lineage I between any of the Nitrospira clusters and other Nitrospira in two WWTPs (Figures 3–5). In a long- AOB, but could not verify this due to the inconsistent term incubation experiment, the Nitrospira from hybridization patterns of the AOB-targeted probes WWTP Vetmed were exposed to different nitrite 6a192 and NEU (Supplementary Text S1). The concentrations and had to compete under these coaggregation of cluster Ig with AOB indicates conditions. Their abundance shifts indicated that that these Nitrospira may prefer high nitrite levels cluster Ie was more competitive in the presence of a found in the close vicinity of AOB microcolonies high nitrite concentration than with a low nitrite (Maixner et al., 2006). Consistently, FISH-MAR level, whereas cluster Id showed the opposite trend showed a strong increase in the CO2 fixation by (Figure 3). The decline of cluster Id in all replicates cluster Ig in the presence of a high nitrite concen- toward the end of the experiment (Figure 3b) tration (1 mM) (Figure 4). However, a specific spatial indicated that these microbes were not well adapted distribution might also reflect other AOB–NOB to the incubation conditions (e.g., the intensity of interactions such as an exchange of excreted aeration that was identical in all incubations) organics or the utilization of siderophores produced and were finally outcompeted by other lineage I by the respective partner (Chain et al., 2003). Nitrospira. This suggests additional ecophysiological Similarly, a negative spatial correlation as observed differences not identified in our study. for cluster Ib and the Ncom1025-targeted AOB Ecological niche partitioning was also shown by (Figure 5c) could indicate for example competition FISH-MAR analyses of three Nitrospira lineage I for oxygen or limited co-factors required by both clusters in WWTP Ingolstadt with respect to carbon groups. Recently, distinct cocorrelation patterns assimilation (Figure 4). Interestingly, all three were described for terrestrial Nitrospira and AOA Nitrospira clusters utilized formate, although to a (Pester et al., 2013). Thus, specific interactions different extent (Figure 4). To the best of our among nitrifiers of different phylogenetic lineages knowledge, this study is the first to demonstrate could be a widespread and interesting phenomenon experimentally the use of formate by Nitrospira. that awaits further analysis. Alternatively, the This feature of Nitrospira could be advantageous positive and negative spatial correlation of close to anoxic niches in sludge flocs or biofilm, Nitrospira clusters Ig and Ib with Ncom1025- where formate may be released by fermenting targeted AOB may be caused by preferences for microorganisms, or in nitrifying bioreactors treating similar or dissimilar environmental conditions sludge liquor from anaerobic digesters. Indeed, the within sludge flocs rather than by specific interac- source reactor of these Nitrospira at WWTP Ingol- tions. This would indicate distinct habitat require- stadt treats sludge liquor. As formate is oxidized to ments of clusters Ia, Ib and Ig. The lack of CO2 by formate dehydrogenase and CO2 is then fixed coaggregation between AOB and lineage II (Lu¨ cker et al., 2010), its use as a carbon source is not Nitrospira contrasts previous results obtained for advantageous compared with autotrophy. However, other WWTPs (Maixner et al., 2006) and could be as electron donor with a much lower redox caused by similar effects as discussed for lineage I potential, formate should be a better energy source clusters (see above) or might even indicate that than nitrite. Nevertheless, clusters Ia and Ig appar- these Nitrospira did not rely on nitrite as energy ently required nitrite (added directly to the medium source. or provided by ammonia oxidation) for carbon In summary, our results show that ecological assimilation from formate (Figure 4), in contrast to niche differentiation has a role for the coexistence cluster Ib that seemed to be highly efficient in using of closely related Nitrospira in WWTPs. The impact formate as carbon and also as energy source. The of other factors, such as microbial predation MAR labelling of each Nitrospira cluster was never (Dolinsˇek et al., 2013), on Nitrospira community completely positive or negative. Instead, different structures remains to be determined. fractions of the counted microcolonies from the same Nitrospira cluster were MAR-positive in the various experiments (Figure 4). This heterogeneity Function of a diverse Nitrospira community may indicate activity differences between micro- The continuous presence of several closely related colonies or further ecophysiological diversification members with different ecophysiology may have at finer phylogenetic scales than resolved by our interesting implications for Nitrospira community FISH approach. function. The resulting more efficient use of Moreover, the three Nitrospira lineage I clusters in the available niche space might improve the WWTP Ingolstadt showed different localization performance of nitrite oxidation, as shown for patterns relative to probe Ncom1025-targeted AOB, other functionally redundant groups and processes indicating niche separation in addition to the (Tilman et al., 1997; Loreau et al., 2001; Cardinale, differential use of formate. Only cluster Ig coaggre- 2011). Furthermore, a diverse Nitrospira commu- gated with these AOB (Figure 5), suggesting that the nity may be advantageous for the stability of nitrite symbiosis among nitrifiers can be selective at the oxidation due to a favorable diversity–stability

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 653 relationship (Yachi and Loreau, 1999; Daims et al., Acknowledgements 2001b; Elmqvist et al., 2003; Tilman et al., 2006; Eisenhauer et al., 2012). With few Nitrospira OTUs We thank Sebastian Lu¨ cker for establishing an ARB database of Nitrospira 16S rRNA genes, David Berry and being highly abundant at a particular time, a large Martin Gruber-Dorninger for help with statistical analyses, diversity of less abundant Nitrospira (Figure 6) may Jan Dolinsˇek, Jeppe Lund Nielsen and Hanna Koch for act as seed bank for compensatory growth after helpful discussions, and Annika Hennig, Christian Bar- disturbances such as changes in the wastewater anyi and Martina Grill for excellent technical assistance. composition in a WWTP or phage attack. As closely We also thank the operators of the WWTPs Ingolstadt and related strains can show high genomic plasticity Vetmed for providing activated sludge samples. This work (e.g., Thompson et al., 2005; Cuadros-Orellana et al., was supported by the Vienna Science and Technology 2007; Denef et al., 2010; Albertsen et al., 2012), a Fund (WWTF, Grant LS09-40 to HD), an ERC Advanced high Nitrospira diversity may also represent a Grant (Nitricare, Grant 294343, to MW) and the Austrian large pool of potentially advantageous genes or Science Fund (FWF, Grant P25231-B21 to HD). alleles that could be exchanged by horizontal transfer within the Nitrospira community (e.g., Cuadros-Orellana et al., 2007; Simmons et al., 2008; Cordero et al., 2012) or homologous recombi- nation (Eppley et al., 2007; Denef and Banfield, References 2012), respectively. Acinas SG, Klepac-Ceraj V, Hunt DE, Pharino C, Ceraj I, Distel DL et al. (2004). Fine-scale phylogenetic architecture of a complex bacterial community. Nature Implications for future nitrification research 430: 551–554. While past studies almost exclusively analyzed Acinas SG, Haverkamp TH, Huisman J, Stal LJ. (2009). Phenotypic and genetic diversification of Pseudanabaena Nitrospira in WWTPs at the level of the whole spp. (cyanobacteria). ISME J 3: 31–46. genus or its major lineages (e.g., Schramm et al., Albertsen M, Hansen LB, Saunders AM, Nielsen PH, 1998; Daims et al., 2001a; Maixner et al., 2006; Park Nielsen KL. (2012). A metagenome of a full-scale and Noguera, 2008), our results imply that a higher microbial community carrying out enhanced biological phylogenetic resolution is needed to understand phosphorus removal. ISME J 6: 1094–1106. the ecophysiological diversification and abundance Almstrand R, Daims H, Persson F, Sorensson F, dynamics of these organisms. Still, it remains to be Hermansson M. (2013). New methods for analysis of determined which units of diversity comprise spatial distribution and coaggregation of microbial ecologically cohesive Nitrospira populations. For populations in complex biofilms. Appl Environ example, all analyzed Nitrospira clusters contained Microbiol 79: 5978–5987. Becraft ED, Cohan FM, Kuhl M, Jensen SI, Ward DM. highly similar but not identical 16S rRNA sequences (2011). Fine-scale distribution patterns of Synechococcus (Figure 1 and Supplementary Table S2). These ecological diversity in microbial mats of Mushroom sequences might represent Nitrospira with further Spring, Yellowstone National Park. Appl Environ functional differentiation or just allelic diversity of Microbiol 77: 7689–7697. the 16S rRNA gene among ecophysiologically Berger SA, Krompass D, Stamatakis A. (2011). Perfor- identical Nitrospira. Moreover, as we analyzed only mance, accuracy, and Web server for evolutionary two relatively highly conserved marker genes coding placement of short sequence reads under maximum for 16S rRNA and nxrB, the detected Nitrospira likelihood. Syst Biol 60: 291–302. diversity may only be the tip of an iceberg. A next Blainey PC. (2013). The future is now: single-cell step could be genome-wide comparative analyses genomics of bacteria and archaea. FEMS Microbiol Rev 37: 407–427. of coexisting Nitrospira complemented by physio- Brown MV, Lauro FM, DeMaere MZ, Muir L, Wilkins D, logical experiments to validate genome-based Thomas T et al. (2012). Global biogeography of SAR11 hypotheses on metabolic functions. With single-cell marine bacteria. Mol Syst Biol 8: 595. genomics (Blainey, 2013) and methods to study Burrell PC, Keller J, Blackall LL. (1998). Microbiology of a single-cell ecophysiology in situ (Wagner et al., nitrite-oxidizing bioreactor. Appl Environ Microbiol 2006; Wagner, 2009), the required tools would in 64: 1878–1883. principle be available. If ammonia oxidizers are also Cardinale BJ. (2011). Biodiversity improves water quality included, nitrifiers in WWTPs can become model through niche partitioning. Nature 472: 86–89. organisms for studying functional and evolutionary Chain P, Lamerdin J, Larimer F, Regala W, Lao V, Land M aspects of bacterial diversity with a high phyloge- et al. (2003). Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemo- netic resolution, also in the context of symbiosis, in lithoautotroph Nitrosomonas europaea. J Bacteriol an easily accessible but complex model ecosystem 185: 2759–2773. (Daims et al., 2006b). Coleman ML, Chisholm SW. (2007). Code and context: Prochlorococcus as a model for cross-scale biology. Trends Microbiol 15: 398–407. Conflict of Interest Connor N, Sikorski J, Rooney AP, Kopac S, Koeppel AF, Burger A et al. (2010). Ecology of speciation in the genus The authors declare no conflict of interest. Bacillus. Appl Environ Microbiol 76: 1349–1358.

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 654 Cordero OX, Wildschutte H, Kirkup B, Proehl S, Ngo L, on the diversity of nitrite-oxidizing bacteria in Hussain F et al. (2012). Ecological populations of agricultural grassland soils. Appl Environ Microbiol bacteria act as socially cohesive units of antibiotic 71: 8323–8334. production and resistance. Science 337: 1228–1231. Fuhrman JA, Steele JA. (2008). Community structure of Cuadros-Orellana S, Martin-Cuadrado AB, Legault B, marine bacterioplankton: patterns, networks, and D’Auria G, Zhaxybayeva O, Papke RT et al. (2007). relationships to function. Aquat Microb Ecol 53: 69–81. Genomic plasticity in prokaryotes: the case of the Good IJ. (1953). The population frequencies of species and square haloarchaeon. ISME J 1: 235–245. the estimation of population parameters. Biometrika Daims H, Bru¨ hl A, Amann R, Schleifer KH, Wagner M. 40: 237–264. (1999). The domain-specific probe EUB338 is insuffi- Haverkamp TH, Schouten D, Doeleman M, Wollenzien U, cient for the detection of all Bacteria: development Huisman J, Stal LJ. (2009). Colorful microdiversity of and evaluation of a more comprehensive probe set. Synechococcus strains (picocyanobacteria) isolated Syst Appl Microbiol 22: 434–444. from the Baltic Sea. ISME J 3: 397–408. Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M. He S, Gall DL, McMahon KD. (2007). ‘Candidatus (2001a). In situ characterization of Nitrospira-like Accumulibacter’ population structure in enhanced nitrite-oxidizing bacteria active in wastewater treat- biological phosphorus removal sludges as revealed ment plants. Appl Environ Microbiol 67: 5273–5284. by polyphosphate kinase genes. Appl Environ Daims H, Purkhold U, Bjerrum L, Arnold E, Wilderer PA, Microbiol 73: 5865–5874. Wagner M. (2001b). Nitrification in sequencing Hicks RE, Amann RI, Stahl DA. (1992). Dual staining of biofilm batch reactors: lessons from molecular natural bacterioplankton with 4’,6-diamidino-2- approaches. Water Sci Technol 43: 9–18. phenylindole and fluorescent oligonucleotide probes Daims H, Stoecker K, Wagner M. (2005). Fluorescence targeting kingdom-level 16S rRNA sequences. Appl in situ hybridization for the detection of prokaryotes. Environ Microbiol 58: 2158–2163. In: Osborn AM, Smith CJ (eds) Advanced Methods in Hunt DE, David LA, Gevers D, Preheim SP, Alm EJ, Molecular Microbial Ecology. Bios-Garland: Abingdon, Polz MF. (2008). Resource partitioning and sympatric UK, pp 213–239. differentiation among closely related bacterioplankton. Daims H, Lu¨ cker S, Wagner M. (2006a). daime, a novel Science 320: 1081–1085. image analysis program for microbial ecology and Juretschko S, Timmermann G, Schmid M, Schleifer KH, biofilm research. Environ Microbiol 8: 200–213. Pommerening-Ro¨ser A, Koops HP et al. (1998). Daims H, Taylor MW, Wagner M. (2006b). Wastewater Combined molecular and conventional analyses of treatment: a model system for microbial ecology. nitrifying bacterium diversity in activated sludge: Trends Biotechnol 24: 483–489. Nitrosococcus mobilis and Nitrospira-like bacteria as Daims H, Wagner M. (2007). Quantification of uncultured dominant populations. Appl Environ Microbiol 64: microorganisms by fluorescence microscopy and 3042–3051. digital image analysis. Appl Microbiol Biotechnol 75: Koeppel A, Perry EB, Sikorski J, Krizanc D, Warner A, 237–248. Ward DM et al. (2008). Identifying the fundamental Denef VJ, Kalnejais LH, Mueller RS, Wilmes P, Baker BJ, units of bacterial diversity: a paradigm shift to Thomas BC et al. (2010). Proteogenomic basis for incorporate ecology into bacterial systematics. ecological divergence of closely related bacteria in Proc Natl Acad Sci USA 105: 2504–2509. natural acidophilic microbial communities. Proc Natl Kunin V, Engelbrektson A, Ochman H, Hugenholtz P. Acad Sci USA 107: 2383–2390. (2010). Wrinkles in the rare biosphere: pyrosequen- Denef VJ, Banfield JF. (2012). In situ evolutionary rate cing errors can lead to artificial inflation of diversity measurements show ecological success of recently estimates. Environ Microbiol 12: 118–123. emerged bacterial hybrids. Science 336: 462–466. Lebedeva EV, Alawi M, Maixner F, Jozsa PG, Daims H, Dolinsˇek J, Lagkouvardos I, Wanek W, Wagner M, Spieck E. (2008). Physiological and phylogenetic Daims H. (2013). Interactions of nitrifying bacteria characterization of a novel lithoautotrophic nitrite- and heterotrophs: identification of a Micavibrio-like oxidizing bacterium, ‘Candidatus Nitrospira bockiana’. putative predator of Nitrospira spp. Appl Environ Int J Syst Evol Microbiol 58: 242–250. Microbiol 79: 2027–2037. Lee N, Nielsen PH, Andreasen KH, Juretschko S, Ehrich S, Behrens D, Lebedeva E, Ludwig W, Bock E. Nielsen JL, Schleifer KH et al. (1999). Combination (1995). A new obligately chemolithoautotrophic, of fluorescent in situ hybridization and micro- nitrite-oxidizing bacterium, Nitrospira moscoviensis autoradiography—a new tool for structure–function sp. nov. and its phylogenetic relationship. Arch analyses in microbial ecology. Appl Environ Microbiol Microbiol 164: 16–23. 65: 1289–1297. Eisenhauer N, Scheu S, Jousset A. (2012). Bacterial Loreau M, Naeem S, Inchausti P, Bengtsson J, Grime JP, diversity stabilizes community productivity. PLoS Hector A et al. (2001). Biodiversity and ecosystem One 7: e34517. functioning: current knowledge and future challenges. Elmqvist T, Folke C, Nystrom M, Peterson G, Bengtsson J, Science 294: 804–808. Walker B et al. (2003). Response diversity, ecosystem Lu¨ cker S, Wagner M, Maixner F, Pelletier E, Koch H, change, and resilience. Front Ecol Environ 1: 488–494. Vacherie B et al. (2010). A Nitrospira metagenome Eppley JM, Tyson GW, Getz WM, Banfield JF. (2007). illuminates the physiology and evolution of globally Genetic exchange across a species boundary in the important nitrite-oxidizing bacteria. Proc Natl Acad archaeal genus ferroplasma. Genetics 177: 407–416. Sci USA 107: 13479–13484. Felsenstein J. (1989). PHYLIP—Phylogeny Inference Pack- Lu¨ cker S, Nowka B, Rattei T, Spieck E, Daims H. (2013). age (Version 3.2). Cladistics 5: 164–166. The genome of Nitrospina gracilis illuminates the Freitag TE, Chang L, Clegg CD, Prosser JI. (2005). metabolism and evolution of the major marine nitrite Influence of inorganic nitrogen management regime oxidizer. Front Microbiol 4: 27.

The ISME Journal Functionally relevant high Nitrospira diversity C Gruber-Dorninger et al 655 Ludwig W, Strunk O, Westram R, Richter L, Meier H, Spieck E, Hartwig C, McCormack I, Maixner F, Wagner M, Yadhukumar et al. (2004). ARB: a software environ- Lipski A et al. (2006). Selective enrichment and ment for sequence data. Nucleic Acids Res 32: molecular characterization of a previously uncultured 1363–1371. Nitrospira-like bacterium from activated sludge. Maixner F, Noguera DR, Anneser B, Stoecker K, Wegl G, Environ Microbiol 8: 405–415. Wagner M et al. (2006). Nitrite concentration Stamatakis AP, Ludwig T, Meier H, Wolf MJ. (2002). influences the population structure of Nitrospira-like AxML: a fast program for sequential and parallel bacteria. Environ Microbiol 8: 1487–1495. phylogenetic tree calculations based on the maximum Malmstrom RR, Coe A, Kettler GC, Martiny AC, likelihood method. Proc IEEE Comput Soc Bioinform Frias-Lopez J, Zinser ER et al. (2010). Temporal Conf 1: 21–28. dynamics of Prochlorococcus ecotypes in the Atlantic Stein LY, Arp DJ. (1998). Loss of ammonia monooxygenase and Pacific oceans. ISME J 4: 1252–1264. activity in Nitrosomonas europaea upon exposure to Ngugi DK, Stingl U. (2012). Combined analyses of the ITS nitrite. Appl Environ Microbiol 64: 4098–4102. loci and the corresponding 16S rRNA genes reveal Stoecker K, Dorninger C, Daims H, Wagner M. (2010). high micro- and macrodiversity of SAR11 populations Double labeling of oligonucleotide probes for fluores- in the Red Sea. PLoS One 7: e50274. cence in situ hybridization (DOPE-FISH) improves Off S, Alawi M, Spieck E. (2010). Enrichment and signal intensity and increases rRNA accessibility. physiological characterization of a novel Nitrospira- Appl Environ Microbiol 76: 922–926. like bacterium obtained from a marine sponge. Thompson JR, Pacocha S, Pharino C, Klepac-Ceraj V, Appl Environ Microbiol 76: 4640–4646. Hunt DE, Benoit J et al. (2005). Genotypic diversity Park HD, Noguera DR. (2008). Nitrospira community within a natural coastal bacterioplankton population. composition in nitrifying reactors operated with Science 307: 1311–1313. two different dissolved oxygen levels. J Microbiol Tilman D, Lehman CL, Thomson KT. (1997). Plant diversity Biotechnol 18: 1470–1474. and ecosystem productivity: theoretical considerations. Pena A, Teeling H, Huerta-Cepas J, Santos F, Yarza P, Proc Natl Acad Sci USA 94: 1857–1861. Brito-Echeverria J et al. (2010). Fine-scale evolution: Tilman D, Reich PB, Knops JM. (2006). Biodiversity genomic, phenotypic and ecological differentiation in and ecosystem stability in a decade-long grassland two coexisting Salinibacter ruber strains. ISME J 4: experiment. Nature 441: 629–632. 882–895. Verbeke TJ, Dumonceaux TJ, Wushke S, Cicek N, Levin DB, Pester M, Rattei T, Flechl S, Grongroft A, Richter A, Sparling R. (2011). Isolates of Thermoanaerobacter Overmann J et al. (2012). amoA-based consensus thermohydrosulfuricus from decaying wood compost phylogeny of ammonia-oxidizing archaea and deep display genetic and phenotypic microdiversity. FEMS sequencing of amoA genes from soils of four different geographic regions. Environ Microbiol 14: 525–539. Microbiol Ecol 78: 473–487. Wagner M, Nielsen PH, Loy A, Nielsen JL, Daims H. Pester M, Maixner F, Berry D, Rattei T, Koch H, Lu¨ cker S et al. (2013). NxrB encoding the beta subunit of (2006). Linking microbial community structure with nitrite oxidoreductase as functional and phylogenetic function: fluorescence in situ hybridization-micro- marker for nitrite-oxidizing Nitrospira. Environ autoradiography and isotope arrays. Curr Opin Biotechnol Microbiol; e-pub ahead of print 10 October 2013; 17: 83–91. doi:10.1111/1462-2920.12300. Wagner M. (2009). Single-cell ecophysiology of microbes Schramm A, de Beer D, Wagner M, Amann R. (1998). as revealed by Raman microspectroscopy or secondary Identification and activities in situ of Nitrosospira ion mass spectrometry imaging. Annu Rev Microbiol and Nitrospira spp. as dominant populations in a 63: 411–429. nitrifying fluidized bed reactor. Appl Environ Microb Watson SW, Bock E, Valois FW, Waterbury JB, Schlosser U. 64: 3480–3485. (1986). Nitrospira marina gen. nov. sp. nov.: a Schramm A, Santegoeds CM, Nielsen HK, Ploug H, chemolithotrophic nitrite-oxidizing bacterium. Arch Wagner M, Pribyl M et al. (1999). On the occurrence Microbiol 144: 1–7. of anoxic microniches, denitrification, and sulfate Woebken D, Lam P, Kuypers MM, Naqvi SW, Kartal B, reduction in aerated activated sludge. Appl Environ Strous M et al. (2008). A microdiversity study of Microbiol 65: 4189–4196. anammox bacteria reveals a novel Candidatus Schramm A, Fuchs BM, Nielsen JL, Tonolla M, Stahl DA. Scalindua phylotype in marine oxygen minimum (2002). Fluorescence in situ hybridization of 16S zones. Environ Microbiol 10: 3106–3119. rRNA gene clones (Clone-FISH) for probe validation Yachi S, Loreau M. (1999). Biodiversity and ecosystem and screening of clone libraries. Environ Microbiol 4: productivity in a fluctuating environment: the 713–720. insurance hypothesis. Proc Natl Acad Sci USA 96: Simmons SL, Dibartolo G, Denef VJ, Goltsman DS, 1463–1468. Thelen MP, Banfield JF. (2008). Population genomic Zo YG, Chokesajjawatee N, Arakawa E, Watanabe H, analysis of strain variation in Leptospirillum group II Huq A, Colwell RR. (2008). Covariability of Vibrio bacteria involved in acid mine drainage formation. cholerae microdiversity and environmental para- PLoS Biol 6: e177. meters. Appl Environ Microbiol 74: 2915–2920.

Supplementary Information accompanies this paper on The ISME Journal website (http://www.nature.com/ismej)

The ISME Journal